Gastroschisis in one twin neonate with extracorporeal liver.

We report a rare case of gastroschisis with extracorporeal liver suspected on late first trimester ultrasound and confirmed with second trimester ultrasound and magnetic resonance imaging in one fetus in a twin pregnancy. Liver herniation is common in omphalocele, a membrane-covered abdominal wall defect associated with other congenital anomalies. However, it is highly uncommon in gastroschisis, an uncovered abdominal wall defect aside of the cord insertion. Presence of liver herniation complicates prenatal differentiation between omphalocele and gastroschisis. The twins were born at 31 weeks' gestation due to preterm labor. The baby was treated with a silo device, followed by biologic mesh and a wound vac with instillation of fluid to prevent desiccation. Ultimately, the baby died of sepsis, with multiorgan failure and polymicrobial infection.

Long-term growth outcomes in neonates diagnosed with necrotizing enterocolitis: a 20-year analysis.

PURPOSE: Limited data exists for longitudinal growth outcomes in neonates with a history of necrotizing enterocolitis (NEC). We aimed to study 20-year growth outcomes in NEC survivors. METHODS: A retrospective matched cohort study included neonates diagnosed with NEC and control subjects matched for birth year, birth weight, and gestational age who had at least one post-discharge follow-up. The primary outcome was growth, measured by length and weight until 20 years. Logistic regression was used to test the change in growth from birth until most recent encounter. RESULTS: Five hundred twenty-seven neonates were included: 294 with NEC, and 233 controls. Sixty-eight percent of NEC cases were Bell's stage I, 25% were stage II, and 7% were stage III. Median gestational age was 29 weeks, and median birth weight was 1237 g. Infants with NEC had a longer NICU stay (p < 0.0001) and increased number of comorbidities (p < 0.0001). Compared to overall and sex-matched controls, infants with NEC had a significantly slower growth rate in terms of weight (p < 0.0068) but not length (p = 0.09). Neither group exhibited failure to thrive. CONCLUSIONS: These results suggest that non-surgical NEC may have a more profound impact on long-term growth than previously considered. TYPE OF STUDY: Retrospective Cohort-Matched Study. LEVEL OF EVIDENCE: Level III.

Appropriate use of total parenteral nutrition in children with perforated appendicitis.

BACKGROUND: Total parenteral nutrition (TPN) is often used in children with perforated appendicitis, despite the absence of clear indications. We assessed the validity of specific clinical indications for initiation of TPN in this patient cohort. METHODS: Data were gathered prospectively on duration of nil per os (NPO) status and TPN use in a cohort of children treated under a perforated appendicitis protocol during a 19-month period. TPN was started in the immediate postoperative period in patients who had generalized peritonitis and severe intestinal dilatation at operation, or later per the discretion of the attending surgeon. At discharge, TPN was considered to have been used appropriately, according to consensus guidelines, if the patient was NPO≥7days or received TPN≥5days. RESULTS: During the study period, TPN was initiated in 31 (25.4%) of 122 patients operated for perforated appendicitis. Sixteen (51.6%) received TPN per operative finding indications and 15 (48.4%) for prolonged ileus. The operative indications demonstrated 47% sensitivity, 86% specificity, a positive predictive value (PPV) of 35%, and a negative predictive value (NPV) of 91%, when adherence to TPN consensus guidelines was considered the gold standard. CONCLUSION: Patients without severe intestinal dilatation and generalized peritonitis at operation should not be placed on TPN in the immediate postoperative period. Refinement of selection criteria is necessary to further decrease inappropriate TPN use in children with perforated appendicitis. TYPE OF STUDY: Diagnostic Test. LEVEL OF STUDY: II.

Experience with peritoneal thermal injury during subcutaneous endoscopically assisted ligation for pediatric inguinal hernia.

BACKGROUND: Subcutaneous endoscopically-assisted ligation (SEAL) for pediatric inguinal hernia repair has gained in popularity although variations in techniques exist. Peritoneal scarring by thermal injury has been described as an adjunct. We explored the hypothesized inverse-correlation between peritoneal scarring and recurrence after SEAL. METHODS: We conducted a single-center retrospective review of all patients <18years old undergoing SEAL between 2010 and 2016 (REB-20172727). Demographics and outcomes were investigated. Univariate and multivariable logistic regressions were performed to evaluate the association between peritoneal scarring and recurrence. RESULTS: We identified 272 patients. Median age was 3years, 35% were female, and 19% were born premature. Median follow-up was 30months, ≥1 visit/patient. Bilaterality was noted in 35%. There were no reported cases of metachronous hernia, vas injury, testicular atrophy or chronic pain, and recurrence rate was 4.6%. Prematurity, unilateral repair, incarceration, and suture-type (Ti-Cron® vs. Ethibond®) had significant correlation with recurrence on univariate analysis (p<0.25). Surgeon experience did not. Peritoneal scarring, performed in 195 cases (72%), was not predictive of recurrence (adjusted OR=0.87, p=0.830) on multivariable analysis. CONCLUSION: The rate of complications with SEAL compares favorably to published data. Thermal injury was not associated with improved recurrence rates. The benefits of peritoneal scarring may not outweigh the risks. LEVEL OF EVIDENCE: III - Retrospective Case-Control Study.

Burn Injury-Associated MHCII+ Immune Cell Accumulation Around Lymphatic Vessels of the Mesentery and Increased Lymphatic Endothelial Permeability Are Blocked by Doxycycline Treatment.

It is theorized that toxic agents are transported from the hyperpermeable gut of burn victims through the lymph, to the systemic circulation, causing global injury. We believe that immune cells respond to leakage of "toxic lymph" following trauma causing the attraction of these cells to the perilymphatic space. To test this, we utilized a model of burn on rats to examine changes in a single immune cell population associated with mesenteric lymphatic dysfunction. We examined the ability of serum from these animals to increase permeability in lymphatic endothelial monolayers and disrupt cellular junctions. We also treated burn animals with doxycycline, an inhibitor of microvascular permeability, and observed the effects on immune cell populations, morphometry, and lymphatic endothelial permeability. Burn injury increased the number of MHCII+ immune cells along the vessel (>50%). The size and shape of these cells also changed significantly following burn injury. Serum from burn animals increased lymphatic endothelial permeability (∼1.5-fold) and induced breaks in VE-cadherin staining. Doxycycline treatment blocked the accumulation of immune cells along the vessel, whereas serum from doxycycline-treated animals failed to increase lymphatic endothelial permeability. The size of cells along the vessel in doxycycline-treated burn animals was not affected, suggesting that the cells already present on the lymphatic vessels still respond to substances in the lymph. These findings suggest that factors produced during burn can induce lymphatic endothelial barrier disruption and lymph produced during traumatic injury can influence the attraction and morphology of immune cell populations along the vessel.

Risk stratification in pediatric perforated appendicitis: Prospective correlation with outcomes and resource utilization.

PURPOSE: Despite a wide spectrum of severity, perforated appendicitis in children is typically considered a single entity in outcomes studies. We performed a prospective cohort study to define a risk stratification system that correlates with outcomes and resource utilization. METHODS: A prospective study was conducted of all children operated for perforated appendicitis between May 2015 and December 2016 at a tertiary free-standing university children's hospital. Surgical findings were classified into one of four grades of perforation: I. localized or contained perforation, II. Contained abscess with no generalized peritonitis, III. Generalized peritonitis with no dominant abscess, IV. Generalized peritonitis with one or more dominant abscesses. All patients were treated on a clinical pathway that involved all points of care from admission to final follow-up. Outcomes and resource utilization measures were analyzed using Fisher's exact test, Kruskal-Wallis test, One-way ANOVA, and logistic regression. RESULTS: During the study period, 122 patients completed treatment, and 100% had documented follow-up at a median of 25days after operation. Grades of perforation were: I, 20.5%; II, 37.7%; III, 10.7%; IV, 31.1%. Postoperative abscesses occurred in 12 (9.8%) of patients, almost exclusively in Grade IV perforations. Hospital stay, duration of antibiotics, TPN utilization, and the incidence of postoperative imaging significantly increased with increasing grade of perforation. CONCLUSION: Outcomes and resource utilization strongly correlate with increasing grade of perforated appendicitis. Postoperative abscesses, additional imaging, and additional invasive procedures occur disproportionately in patients who present with diffuse peritonitis and abscess formation. The current stratification allows risk-adjusted outcome reporting and appropriate assignment of resource burden. LEVEL OF EVIDENCE: I (Prognosis Study).

Standardization of care for pediatric perforated appendicitis improves outcomes.

BACKGROUND: The treatment of perforated appendicitis in children is characterized by significant variability in care, morbidity, resource utilization, and outcomes. We prospectively studied how minimization of care variability affects outcomes. METHODS: A clinical pathway for perforated appendicitis, in use for three decades, was further standardized in May 2015 by initiation of a disease severity classification, refinement of discharge criteria, standardization of the operation, and establishment of criteria for use of postoperative total parenteral nutrition, imaging, and invasive procedures. Prospective evaluation of all children treated for 20months on the new fully standardized protocol was conducted and compared to a retrospective cohort treated over 58months prior to standardization. Differences between outcomes before and after standardization were analyzed using regression analysis techniques to adjust for disease severity. RESULTS: Median follow-up time post discharge was 25 and 14days in the post- and prestandardization groups, respectively. Standardization significantly reduced postoperative abscess (9.8% vs. 17.4%, p=0.001) and hospital stay (p=0.002). Standardization reduced the odds of developing a postoperative abscess by four fold. CONCLUSION: Minimizing variability of care at all points in the treatment of perforated appendicitis significantly improves outcomes. TYPE OF STUDY: Prospective Cohort Study. LEVEL OF EVIDENCE: Level II.

Variability of diagnostic approach, surgical technique, and medical management for children with biliary atresia in Canada - Is it time for standardization?

BACKGROUND: The Canadian 4-year native liver survival rate for biliary atresia (BA) after Kasai Portoenterostomy (KP) is 39%. The Canadian Biliary Atresia Registry (CBAR) was used to examine variability of surgical and medical management of BA. METHODS: Gastroenterologists and surgeons in all 14 Canadian pediatric tertiary centers were invited to complete an online survey of their BA management practices. RESULTS: Of gastroenterologists, diagnostic procedures included liver biopsy (92%), HIDA scan (58%), and percutaneous cholangiogram (46%). Surgeons reported Roux-en-Y lengths of 20-50cm with 78% avoiding diathermy at the portal plate; 16% performed laparoscopic exploration, but none laparoscopic KP. Postoperative corticosteroids and antibiotics were used by 24% and 85% of gastroenterologists, respectively, with similar rates for surgeons. At discharge, gastroenterologists prescribed oral antibiotics (80%), and ursodeoxycholic acid (95%), while surgeons reported lower rates (62% and 55%). Considerable variation existed in follow-up monitoring. No center had a standard protocol for evaluating suspected cholangitis. There was a lack of consensus for defining failed KP and referral criteria for transplant evaluation. CONCLUSION: In Canada, treatment of BA is not centralized, and there is variability in diagnostic approaches and management. Collaboration through CBAR will allow for implementation and evaluation of standardized surgical and medical management with a goal to improve outcomes. LEVEL OF EVIDENCE: Survey study. Level IV evidence.

Tissue inhibitor of metalloproteinase-2 inhibits burn-induced derangements and hyperpermeability in microvascular endothelial cells.

BACKGROUND: Burns induce microvascular hyperpermeability. We hypothesize that this occurs partly through an imbalance between matrix metalloproteinases (MMPs) and endogenous MMP inhibitors such as tissue inhibitors of metalloproteinases (TIMPs), and that such derangements can be attenuated with the use of TIMP-2. METHOD: Rats underwent either sham or burn: serum and tissue were collected. Western blot was used to examine MMP-9 and TIMP-2 levels and MMP activity was assayed from lung tissue. Rat lung microvascular endothelial cells were used to assess monolayer permeability and evaluate the adherens junction proteins ß-catenin, vascular endothelial cadherin and filamentous actin after exposure to burn serum ± TIMP-2. RESULTS: Lung tissue from burn animals showed increased MMP activity, decreased levels of TIMP-2, and no difference in levels of active MMP-9 in burn vs control groups. Burn serum increased monolayer permeability, damaged adherens junction proteins, and incited actin stress fiber formation; TIMP-2 attenuated these derangements. CONCLUSIONS: Burns may lower TIMP-2 levels and increase MMP activity and that TIMP-2 application in vitro may attenuate burn-induced hyperpermeability and decreases damage to endothelial structural proteins. These links warrant further investigation.

Doxycycline Attenuates Lipopolysaccharide-Induced Microvascular Endothelial Cell Derangements.

INTRODUCTION: Lipopolysaccharide (LPS) is known to induce vascular derangements. The pathophysiology involved therein is unknown, but matrix metalloproteinases (MMPs) may be an important mediator. We hypothesized that in vitro LPS provokes vascular permeability, damages endothelial structural proteins, and increases MMP activity; that in vivo LPS increases permeability and fluid requirements; and that the MMP inhibitor doxycycline mitigates such changes. METHODS: Rat lung microvascular endothelial cells were divided into four groups: control, LPS, LPS plus doxycycline, and doxycycline. Permeability, structural proteins ß-catenin and Filamentous-actin, and MMP-9 activity were examined. Sprauge Dawley rats were divided into sham, IV LPS, and IV LPS plus IV doxycycline groups. Mesenteric postcapillary venules were observed. Blood pressure was measured as animals were resuscitated and fluid requirements were compared. Statistical analysis was conducted using Student's t-test and ANOVA. RESULTS: In vitro LPS increased permeability, damaged adherens junctions, induced actin stress fiber formation, and increased MMP-9 enzyme activity. In vivo, IV LPS administration induced vascular permeability. During resuscitation, significantly more fluid was necessary to maintain normotension in the IV LPS group. Doxycycline mitigated all derangements observed. CONCLUSIONS: We conclude that LPS increases permeability, damages structural proteins, and increases MMP-9 activity in endothelial cells. Additionally, endotoxemia induces hyperpermeability and increases the amount of IV fluid required to maintain normotension in vivo. Doxycycline mitigates such changes both in vitro and in vivo. Our findings illuminate the possible role of matrix metalloproteinases in the pathophysiology of lipopolysaccharide-induced microvascular hyperpermeability and pave the way for better understanding and treatment of this process.

A standard handoff improves cardiac surgical patient transfer: operating room to intensive care unit.

BACKGROUND: Patient handoffs are high-risk times associated with sentinel events. Effective handoff processes may enhance patient safety and team member communication. This study assesses the impact of a standardized protocol for handoffs from the cardiac surgery operating room to intensive care unit (ICU). METHODS: Using a prospective pre-post study design, a formalized handoff process was developed including critical handoff elements and a standardized handoff procedure, script, and checklist. Data were collected from 60 handoff observations (30 pre and 30 post), evaluating 52 unique parameters, and survey of providers on perspectives of the handoff process. Results were compared by chi-square test, two sample t-test, or nonparametric Mann-Whitney test. Statistical significance was defined as P ≤ .05. RESULTS: Provider's perspectives showed improved satisfaction with the standardized handoff process through improved responses in 19 of 22 survey items (P < .001). Median time until ventilator connection, ICU monitor transfer, first cardiac index, and chest radiograph were reduced after implementation. Completion of handoff process components also improved after implementation for 36 of 47 nontime parameters. CONCLUSIONS: A standard checklist-driven handoff process can dramatically improve key data transmission and reduce time of critical patient care steps during the high-risk period of patient handoff in a cardiac surgical ICU.

Tumor necrosis factor-alpha-induced microvascular endothelial cell hyperpermeability: role of intrinsic apoptotic signaling

Tumor necrosis factor-alfa (TNF-alfa), a pro-apoptotic cytokine, is involved in vascular hyperpermeability, tissue edema, and inflammation. We hypothesized that TNF-alfa induces microvascular hyperpermeability through the mitochondria-mediated intrinsic apoptotic signaling pathway. Rat lung microvascular endothelial cells grown on Transwell inserts, chamber slides, or dishes were treated with recombinant TNF-alfa (10 ng/ml) in the presence or absence of a caspase-3 inhibitor, Z-DEVD-FMK (100 μM). Fluorescein isothiocyanate (FITC)-albumin (5 mg/ml) was used as a marker of monolayer permeability. Mitochondrial reactive oxygen species (ROS) was determined using dihydrorhodamine 123 and mitochondrial transmembrane potential using JC-1. The adherens junction integrity and actin cytoskeletal organization were studied using β-catenin immunofluorescence and rhodamine phalloidin, respectively. Caspase-3 activity was measured fluorometrically. The pretreatment with Z-DEVD-FMK (100 μM) attenuated TNF-alfa-induced (10 ng/ml) disruption of the adherens junctions, actin stress fiber formation, increased caspase-3 activity, and monolayer hyperpermeability (p < 0.05). TNF-alfa (10 ng/ml) treatment resulted in increased mitochondrial ROS formation and decreased mitochondrial transmembrane potential. Intrinsic apoptotic signaling-mediated caspase-3 activation plays an important role in regulating TNF-α-induced endothelial cell hyperpermeability

Tumor necrosis factor-α-induced microvascular endothelial cell hyperpermeability: role of intrinsic apoptotic signaling.

Tumor necrosis factor-α (TNF-α), a pro-apoptotic cytokine, is involved in vascular hyperpermeability, tissue edema, and inflammation. We hypothesized that TNF-α induces microvascular hyperpermeability through the mitochondria-mediated intrinsic apoptotic signaling pathway. Rat lung microvascular endothelial cells grown on Transwell inserts, chamber slides, or dishes were treated with recombinant TNF-α (10 ng/ml) in the presence or absence of a caspase-3 inhibitor, Z-DEVD-FMK (100 µM). Fluorescein isothiocyanate (FITC)-albumin (5 mg/ml) was used as a marker of monolayer permeability. Mitochondrial reactive oxygen species (ROS) was determined using dihydrorhodamine 123 and mitochondrial transmembrane potential using JC-1. The adherens junction integrity and actin cytoskeletal organization were studied using ß-catenin immunofluorescence and rhodamine phalloidin, respectively. Caspase-3 activity was measured fluorometrically. The pretreatment with Z-DEVD-FMK (100 µM) attenuated TNF-α-induced (10 ng/ml) disruption of the adherens junctions, actin stress fiber formation, increased caspase-3 activity, and monolayer hyperpermeability (p < 0.05). TNF-α (10 ng/ml) treatment resulted in increased mitochondrial ROS formation and decreased mitochondrial transmembrane potential. Intrinsic apoptotic signaling-mediated caspase-3 activation plays an important role in regulating TNF-α-induced endothelial cell hyperpermeability.

Melatonin inhibits thermal injury-induced hyperpermeability in microvascular endothelial cells.

BACKGROUND: Burns induce systemic inflammatory reactions and vascular hyperpermeability. Breakdown of endothelial cell adherens junctions is integral in this process, and reactive oxygen species (ROS) and proteolytic enzymes such as matrix metalloproteinase-9 (MMP-9) play pivotal roles therein. Outside trauma, melatonin has shown to exhibit anti-MMP activity and to be a powerful antioxidant. Consequently, we hypothesized that burn-induced junctional damage and hyperpermeability could be attenuated with melatonin. METHODS: Sprague-Dawley rats were assigned to sham or burn groups. Fluorescein isothiocyanate-bovine albumin was administered intravenously. Venules were examined with intravital microscopy; fluorescence intensities were measured intravascularly and extravascularly. Serum was collected. Rat lung microvascular endothelial cells were grown as monolayers and divided into four groups: sham serum and burn serum with and without melatonin pretreatment. Fluorescein isothiocyanate-bovine albumin flux was measured. Immunofluorescence for adherens junction proteins and staining for actin were performed, and images were captured. Cells were grown on 96 well plates, and ROS species generation following application of burn and sham serum was analyzed with and without melatonin. Statistical analysis was conducted with the Student's t test. RESULTS: Intravital microscopy data revealed an increase in vascular hyperpermeability following burn (p < 0.05). Monolayer permeability was increased with burn serum (p < 0.05); this was attenuated with melatonin (p < 0.05). Immunofluorescence showed damage of rat lung microvascular endothelial cell adherens junctions with burn serum exposure, and melatonin restored integrity. Rhodamine phalloidin staining showed filamentous actin stress fiber formation after burn serum application, and melatonin decreased this. Burn serum significantly increased ROS species generation (p < 0.05), and melatonin negated this (p < 0.05). CONCLUSION: Burns damage endothelial adherens junctions and induce microvascular hyperpermeability; melatonin attenuates this process. This insight into the mechanisms of burn-induced fluid leak suggests the role of ROS and MMP-9 but more importantly hints at the possibility of new treatments to combat vascular hyperpermeability in burns.

Reactive oxygen species-caspase-3 relationship in mediating blood-brain barrier endothelial cell hyperpermeability following oxygen-glucose deprivation and reoxygenation.

OBJECTIVE: Microvascular hyperpermeability that occurs due to breakdown of the BBB is a major contributor of brain vasogenic edema, following IR injury. In microvascular endothelial cells, increased ROS formation leads to caspase-3 activation following IR injury. The specific mechanisms, by which ROS mediates microvascular hyperpermeability following IR, are not clearly known. We utilized an OGD-R in vitro model of IR injury to study this. METHODS: RBMEC were subjected to OGD-R in presence of a caspase-3 inhibitor Z-DEVD, caspase-3 siRNA or an ROS inhibitor L-AA. Cytochrome c levels were measured by ELISA and caspase-3 activity was measured fluorometrically. TJ integrity and cytoskeletal assembly were studied using ZO-1 immunofluorescence and rhodamine phalloidin staining for f-actin, respectively. RESULTS: OGD-R significantly increased monolayer permeability, ROS formation, cytochrome c levels, and caspase-3 activity (p < 0.05) and induced TJ disruption and actin stress fiber formation. Z-DEVD, L-AA and caspase-3 siRNA significantly attenuated OGD-R-induced hyperpermeability (p < 0.05) while only L-AA decreased cytochrome c levels. Z-DEVD and L-AA protected TJ integrity and actin cytoskeletal assembly. CONCLUSIONS: These results suggest that OGD-R-induced hyperpermeability is ROS and caspase-3 dependent and can be regulated by their inhibitors.

Doxycycline attenuates burn-induced microvascular hyperpermeability.

BACKGROUND: Burns induce systemic microvascular hyperpermeability resulting in shock, and if untreated, cardiovascular collapse. Damage to the endothelial cell adherens junctional complex plays an integral role in the pathophysiology of microvascular hyperpermeability. We hypothesized that doxycycline, a known inhibitor of matrix metalloproteinases (MMPs), could attenuate burn-induced adherens junction damage and microvascular hyperpermeability. METHODS: Male Sprague-Dawley rats were divided into sham, burn, and burn + doxycycline (n = 5). The experimental groups underwent a 30% total body surface area full-thickness burn. Fluorescein isothiocyanate-albumin was administered intravenously. Mesenteric postcapillary venules were examined with intravital microscopy to determine flux of albumin from the intravascular space to the interstitium. Fluorescence intensity was compared between the intravascular space to the interstitium at 30, 60, 80, 100, 120, 140, 160, and 180 minutes after burn. Parallel experiments were performed in which rat lung microvascular endothelial cells were treated with sera from sham or burn animals as well as separate groups pretreated with either doxycycline or a specific inhibitor of MMP-9. Monolayer permeability was determined by fluorescein isothiocyanate albumin-flux across Transwell plates and immunofluorescense staining for the adherens junction protein ß-catenin was performed. Western blot and gelatin zymography were performed to assess MMP-9 level and activity. RESULTS: MMP-9 levels were increased after burn. Monolayer permeability was significantly increased with burn serum treatment; this was attenuated with doxycycline as well as the specific MMP-9 inhibitor (p < 0.05). Damage of the endothelial cell adherens junction complex was induced by serum from burned rats, and doxycycline restored the integrity of the adherens junction similar to the MMP-9 inhibitor. Intravital microscopy revealed microvascular hyperpermeability after burn; this was attenuated with doxycycline (p < 0.05). CONCLUSION: Burns induce microvascular hyperpermeability via endothelial adherens junction disruption associated with MMP-9, and this is attenuated with doxycycline.

Regulation of tumor necrosis factor-α-induced microvascular endothelial cell hyperpermeability by recombinant B-cell lymphoma-extra large.

BACKGROUND: Tumor necrosis factor-α (TNF-α), a cytotoxic cytokine, induces endothelial cell barrier dysfunction and microvascular hyperpermeability, leading to tissue edema, a hallmark of traumatic injuries. The objective of the present study was to determine whether B-cell lymphoma-extra large (Bcl-xL), an antiapoptotic protein, would regulate and protect against TNF-α-mediated endothelial cell barrier dysfunction and microvascular hyperpermeability. METHODS: Rat lung microvascular endothelial cells were grown as monolayers on Transwell membranes, and fluorescein isothiocyanate-bovine albumin flux (5 mg/mL) across the monolayer was measured fluorometrically to indicate changes in monolayer permeability. The rat lung microvascular endothelial cell adherens junctional integrity and actin cytoskeleton was studied using ß-catenin immunofluorescence and rhodamine phalloidin dye, respectively. Pretreatment of caspase-8 inhibitor (Z-IETD-FMK, 100 µM) for 1 hour and transfection of Bcl-2-homology domain 3-interacting domain death agonist small interfering RNA (10 µM) for 48 hours were performed to study their respective effects on TNF-α-induced (10 ng/mL; 1-hour treatment) monolayer permeability. Recombinant Bcl-xL protein (2.5 µg/ml) was transfected in rat lung microvascular endothelial cells for 1 hour, and its effect on permeability was demonstrated using a permeability assay. Caspase-3 activity was assayed fluorometrically. RESULTS: Z-IETD-FMK pretreatment protected the adherens junctions and decreased TNF-α-induced monolayer hyperpermeability. Bcl-2-homology domain 3-interacting domain death agonist small interfering RNA transfection attenuated the TNF-α-induced increase in monolayer permeability. Recombinant Bcl-xL protein showed protection against TNF-α-induced actin stress fiber formation, an increase in caspase-3 activity, and monolayer hyperpermeability. CONCLUSIONS: Our results have demonstrated the protective effects of recombinant Bcl-xL protein against TNF-α-induced endothelial cell adherens junction damage and microvascular endothelial cell hyperpermeability. These findings support the potential for Bcl-xL-based drug development against microvascular hyperpermeability and tissue edema.

Tumor necrosis factor-related apoptosis-inducing ligand promotes microvascular endothelial cell hyperpermeability through phosphatidylinositol 3-kinase pathway.

BACKGROUND: Microvascular hyperpermeability that occurs in hemorrhagic shock and burn trauma is regulated by the apoptotic signaling pathway. We hypothesized that tumor necrosis factor-α (TNF-α)-related apoptosis-inducing ligand (TRAIL) would promote hyperpermeability directly or by interacting with other signaling pathways. METHODS: Rat lung microvascular endothelial cells (RLMECs) grown on Transwell membranes (Corning Life Sciences, Lowell, MA) were treated with recombinant human TRAIL (10, 50, and 100 ng/mL) for 6 hours or TRAIL (100 ng/mL) + LY294002 (a PI3K inhibitor; 20 µmol/L), Z-DEVD-FMK (a caspase-3 inhibitor; 10 µmol/L), or the inhibitors alone. Fluorescein isothiocyanate (FITC)-albumin flux was an indicator of permeability. Caspase-3 activity was measured fluorometrically. Adherens junction integrity was studied using ß-catenin immunofluorescence. RESULTS: TRAIL + LY294002, but not TRAIL alone, induced monolayer hyperpermeability (P < .05), and caspase-3 activity (P < .05), and disrupted the adherens junctions. Z-DEVD-FMK attenuated hyperpermeability and protected the adherens junctions. CONCLUSIONS: TRAIL-induced microvascular hyperpermeability is phosphatidylinositol 3-kinase (PI3K)-dependent and may be mediated by caspase-3 cleavage of the endothelial adherens junctional complex.

Inhibition of Fas-Fas ligand interaction attenuates microvascular hyperpermeability following hemorrhagic shock.

Hemorrhagic shock (HS)-induced microvascular hyperpermeability poses a serious challenge in the management of trauma patients. Microvascular hyperpermeability occurs mainly because of the disruption of endothelial cell adherens junctions, where the "intrinsic" apoptotic signaling plays a regulatory role. The purpose of this study was to understand the role of the "extrinsic" apoptotic signaling molecules, particularly Fas-Fas ligand interaction in microvascular endothelial barrier integrity. Rat lung microvascular endothelial cells (RLMECs) were exposed to HS serum in the presence or absence of the Fas ligand inhibitor, FasFc. The effect of HS serum on Fas receptor and Fas ligand expression on RLMECs was determined by flow cytometry. Endothelial cell permeability was determined by monolayer permeability assay and the barrier integrity by ß-catenin immunofluorescence. Mitochondrial reactive oxygen species formation was determined using dihydrorhodamine 123 probe by fluorescent microscopy. Mitochondrial transmembrane potential was studied by fluorescent microscopy as well as flow cytometry. Caspase 3 enzyme activity was assayed fluorometrically. Rat lung microvascular endothelial cells exposed to HS serum showed increase in Fas receptor and Fas ligand expression levels. FasFc treatment showed protection against HS serum-induced disruption of the adherens junctions and monolayer hyperpermeability (P < 0.05) in the endothelial cells. Pretreatment with FasFc also decreased HS serum-induced increase in mitochondrial reactive oxygen species formation, restored HS serum-induced drop in mitochondrial transmembrane potential, and reduced HS serum-induced caspase 3 activity in RLMECs. These findings open new avenues for drug development to manage HS-induced microvascular hyperpermeability by targeting the Fas-Fas ligand-mediated pathway.

Laparoscopic cholecystectomy in obese and non-obese children.

BACKGROUND: Obesity is an increasing problem in the pediatric population. Despite abundant data on the impact of obesity in adults, little data exist that examines the impact of obesity on surgical outcomes in children. We reviewed our experience with laparoscopic cholecystectomy to evaluate the impact of obesity. METHODS: We performed a retrospective chart review of patients who underwent laparoscopic cholecystectomy between September, 2000 and June, 2009. Demographics, indication, length of operation, length of stay, and complications were examined. Body mass index (BMI) was calculated and BMI percentage according to gender and age was determined. RESULTS: There were 312 patients identified, 150 patients were normal weight (BMI less than 85%), 65 patients were overweight (BMI = 85%-95%), and 97 patients were obese (BMI > 95%). The mean age of the patients was 14 y (range 0-20), and 76% were female. The overweight and obese groups had more females (P = 0.022 and P = 0.0016) and the obese group was older (P = 0.0003). No differences were found between the groups in the indication for cholecystectomy. There was no difference in operative time, length of stay, or complications between normal weight patients and overweight or obese patients. CONCLUSION: Despite the known surgical challenges with overweight patients, laparoscopic cholecystectomy is a safe and equally beneficial procedure in overweight children.